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Anti-HE4 nano antibody 1A8 and application thereof

A nanobody and antibody technology, applied in the field of immunology, can solve the problems of specific antigen-antibody binding reaction, difficulty in fully identifying antigenic determinants, affecting detection efficiency, etc., and achieve the effect of excellent detection performance.

Active Publication Date: 2021-06-29
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the small molecular weight of HE4, it is difficult for conventional antibodies to fully recognize some antigenic determinants hidden in the gap or cavity. If the antibody recognizes the epitope too single or the sites are too close or overlapping, it will lead to specific antigen-antibody The binding reaction is affected, which seriously affects the detection efficiency

Method used

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  • Anti-HE4 nano antibody 1A8 and application thereof
  • Anti-HE4 nano antibody 1A8 and application thereof
  • Anti-HE4 nano antibody 1A8 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Construction and screening of Nanobody phage display library

[0031] 1.1 Immunity of alpacas

[0032] A healthy adult alpaca was selected, and the recombinant HE4 antigen (GenBank: AAO52683.1, constructed on the pCDNA3.1 vector through HindⅢ and EcoRI restriction sites, was expressed by human 293 cells using conventional molecular biology techniques) Mix with Freund's adjuvant at a ratio of 1:1, and immunize alpacas with 6-7 μg / kg subcutaneous injection at multiple points on the back, and immunize four times with an interval of 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0033] 1.2 Isolation of camel-derived lymphocytes

[0034] Lymphocytes were separated from the collected alpaca peripheral blood using the Camel Peripheral Blood Lymphocyte Separation Solution Kit (Tianjin Haoyang Company, Cat. No. LTS1076). 7 Add 1ml RNA isolation reagent to each living cell, take 1ml for RNA ex...

Embodiment 2

[0063] Example 2. Preparation of Nanobody 1A8

[0064] 2.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL21(DE3)

[0065] Inoculate 5 ml of fresh LB-A medium at a ratio of 1:1000 to inoculate the original strain TG1 Glycerobacterium containing Nanobody nucleic acid, and culture overnight at 37°C and 200 rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the instructions. After verification, transform 1 μl of the above plasmid into 100 μl competent cells, mix gently, place on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and cool in an ice bath for 3 minutes. Add 600 μl LB medium to the centrifuge tube, shake and incubate at 37°C for 60 minutes. Take 100 μl of the supernatant, spread it on the LB-A plate with a triangular spreader, and culture it upside down at 37°C overnight.

[0066] 2.2 Induced expression of nanobodies

[0067] The abov...

Embodiment 3

[0070] Example 3. Determination of the affinity and activity of nanobodies and antigens

[0071] 3.1 Chip antigen coupling

[0072] The antigen was formulated into a 20 μg / ml working solution with different pH sodium acetate buffers (pH 5.5, pH 5.0, pH 4.5, pH 4.0), and a 50 mM NaOH regeneration solution was prepared at the same time, and the Biacore T100 protein interaction analysis system instrument was used to The template method is used to analyze the electrostatic binding between the antigen and the surface of the chip (GE company) under different pH conditions, and the signal increase amount reaches 5 times RL as the standard, select the most suitable neutral pH system and adjust it as needed The antigen concentration was used as the condition during coupling. The chip was coupled according to the template method that comes with the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the designed...

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Abstract

The invention discloses an anti-HE4 nano antibody, the nano antibody has three unique complementary determining regions CDR1, CDR2 and CDR3, the invention also provides an expression vector containing a encoding sequence of a variable region of the nano antibody, a host cell containing the expression vector, and an application of the nano antibody in detection of HE4 content in serum. The nano antibody provided by the invention has specific recognition and binding ability to HE4, the affinity of the nano antibody can reach 1.009 E-09, and the nano antibody has a unique antigenic determinant recognition site and can obtain an excellent detection effect in HE4 serum detection by using a double-antibody sandwich method.

Description

technical field [0001] The invention discloses a nanobody and belongs to the field of immunology. Background technique [0002] Epithelial ovarian cancer (Endometrioid Ovarian Cancer, EOC) is a common malignant tumor of female reproductive organs, and its incidence rate ranks third among female malignant tumors. rate ranks first. Human epididymis protein (HE4) is a new ovarian cancer-specific tumor marker, which belongs to whey acidic protein family (WAP), and is a secreted small molecule glycoprotein encoded by WFDC2 gene. The molecular weight is about 13kDa, and it exists in the form of 25kDa glycosylation after maturity. It is mainly overexpressed in ovarian epithelial cancer and endometrial cancer, and its serum level is significantly more sensitive than CA125 in predicting ovarian cancer, especially in the early stage. The critical value can reach 150pmol / L. Lu Renquan et al. (Evaluation of the Clinical Application of HE4 in the Diagnosis and Treatment of Ovarian Can...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N9/16C12N15/13C12N15/70C12N1/21G01N33/68G01N33/574C12R1/19
CPCC07K16/3069C12N9/16C12N15/70G01N33/57449G01N33/57488G01N33/689C12Y301/03001C07K2317/22C07K2317/565C07K2317/569C07K2317/92C07K2319/00
Inventor 宋海鹏刘原源于建立蒋立仲王准曹慧古一李飞张霞
Owner 深圳市国创纳米抗体技术有限公司
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